CN104991028B - The reduction method of fixedness buffer salt content in LC MS testers - Google Patents
The reduction method of fixedness buffer salt content in LC MS testers Download PDFInfo
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- CN104991028B CN104991028B CN201510463558.8A CN201510463558A CN104991028B CN 104991028 B CN104991028 B CN 104991028B CN 201510463558 A CN201510463558 A CN 201510463558A CN 104991028 B CN104991028 B CN 104991028B
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- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 4
- NPYPAHLBTDXSSS-UHFFFAOYSA-N Potassium ion Chemical compound [K+] NPYPAHLBTDXSSS-UHFFFAOYSA-N 0.000 description 4
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- QEEAPRPFLLJWCF-UHFFFAOYSA-K potassium citrate (anhydrous) Chemical compound [K+].[K+].[K+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O QEEAPRPFLLJWCF-UHFFFAOYSA-K 0.000 description 4
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- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
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- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 description 2
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- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 2
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- KSMRODHGGIIXDV-YFKPBYRVSA-N N-acetyl-L-glutamine Chemical compound CC(=O)N[C@H](C(O)=O)CCC(N)=O KSMRODHGGIIXDV-YFKPBYRVSA-N 0.000 description 1
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Landscapes
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The application is related to a kind of reduction method of fixedness buffer salt content in LC MS testers.Specifically, the application is related to a kind of method of fixedness buffer salt content in reduction liquid chromatogram eluting fraction in test for LC Mass, and this method comprises the following steps：(1) detectable substance is configured to solution, the solution is injected into liquid chromatograph to be eluted, the object eluting fraction for being ready to use in Mass Spectrometer Method is collected from liquid chromatography eluant；(2) eluting fraction is made to be sufficiently mixed with auxiliary agent；(3) make gained mixed liquor separate suspension and clear liquid by way of centrifuging or filtering, divide and take clear liquid；With optional (4) are concentrated gained clear liquid, are produced.The application method can be effectively reduced the buffer salt content in high performance liquid chromatography eluent, so that the eluent is reduced to mass spectrometric loss when for follow-up mass spectroscopy.The present invention uses simple processing method, it is possible to achieve fixedness buffer salinity is substantially reduced in the LC eluting fractions of LC MS methods, and target substance will not be lost.
Description
Technical Field
The application belongs to the technical field of analytical chemistry, relates to related operation in a liquid chromatography-mass spectrometry method, and particularly relates to a method for reducing the content of buffer salt in a test object by a liquid chromatography-mass spectrometry (LC-MS) method. The method can effectively reduce the content of buffer salt in the high performance liquid chromatography eluent, so that the loss of a mass spectrometer is reduced when the eluent is used for subsequent mass spectrometry. The invention adopts a simple processing method, can realize the great reduction of the concentration of the non-volatile buffer salt in the LC elution fraction of the LC-MS method, and can not lose the target substance.
Background
Mass spectrometry is an analysis method for measuring the mass-to-charge ratio (mass-to-charge ratio) of ions, and its basic principle is to ionize each component in a sample in an ion source to generate charged ions with different charge-to-mass ratios, and the charged ions are accelerated by an electric field to form an ion beam, which enters a mass analyzer. In the mass analyzer, the mass is determined by dispersing the generated opposite velocities by an electric field and a magnetic field, and focusing them to obtain mass spectra.
The mass spectrometry is an identification technology, and plays an important role in the identification of organic molecules. It can quickly and accurately measure the molecular weight of biological macromolecular, so that the proteome research can be deeply conducted from protein identification to high-grade structure research and interaction research between various proteins.
With the development of mass spectrometry, the application field of mass spectrometry is wider and wider. Because the mass spectrometry has the advantages of high sensitivity, less sample consumption, high analysis speed, simultaneous separation and identification and the like, the mass spectrometry technology is widely applied to various fields of chemistry, chemical engineering, environment, energy, medicine, sports medicine, criminal science and technology, life science, material science and the like.
Mass spectrometers are widely available and have different application characteristics for different instruments, and generally, a sample that can be vaporized at about 300 ℃ can be preferentially analyzed by gas chromatography-mass spectrometry (GC-MS), because GC-MS uses an EI source, and mass spectrometry information is obtained, so that library search can be performed. The separation effect of the capillary column is also good. If the material can not be vaporized at about 300 ℃, LC-MS analysis is needed, and molecular weight information is mainly obtained at the time, and some structural information can be obtained if the material is a tandem mass spectrum. If the biomacromolecule is a biomacromolecule, the molecular weight information is mainly obtained by mainly utilizing LC-MS and MALDI-TOF analysis. For protein samples, the amino acid sequence can also be determined. The resolution of a mass spectrometer is an important technical index, and a high-resolution mass spectrometer can provide a compound composition formula, which is very important for structural determination. The double-focusing mass spectrometer, the Fourier transform mass spectrometer, the time-of-flight mass spectrometer with the reflector and the like have high resolution functions.
Mass spectrometry has certain requirements on the sample. The sample for GC-MS analysis should be an organic solution, the organic matter in the aqueous solution can not be generally determined, and extraction separation is required to be carried out to obtain the organic solution, or a headspace sampling technology is adopted. Some compounds are too polar and are easily decomposed during heating, for example, organic acid compounds, in which case esterification treatment is performed to convert the acid into an ester, and GC-MS analysis is performed, and the structure of the acid can be estimated from the analysis result. If the sample cannot be vaporized nor esterified, only LC-MS analysis can be performed. For polar samples, an ESI source is generally used, and for non-polar samples, an APCI source is used.
A liquid chromatography-Mass Spectrometer (liquid chromatography Mass Spectrometer), abbreviated as LC-MS, is a device for combining liquid chromatography and Mass spectrometry. The method combines the separation capability of a liquid chromatograph for effectively separating heat instability and high-boiling point compounds with the strong component identification capability of a mass spectrometer. Is an effective means for separating and analyzing complex organic mixtures.
Chromatography-mass spectrometry is one of the most important analytical methods for separation and identification of the current generation. The advantages of chromatography are that the separation capacity of chromatography provides the most effective choice for separating mixtures, but the chromatographic method has difficulty in obtaining structural information, and the method mainly relies on the comparison with standard samples to realize the presumption of the structure of an unknown substance; the method is weak in structural analysis of complex mixed unknowns; the detection of non-uv absorbing compounds and qualitative analysis of large amounts of unknown compounds on conventional uv detectors also relies on other means. Mass spectrometry provides abundant structural information, and the sample amount is the least used in several spectroscopic methods, but the sample needs to be pretreated (purified and separated), and the process is complex and takes long time. For a long time, many techniques have been developed to solve the weaknesses of the two technologies, and the chromatography-mass spectrometry technology is one of the most promising technologies. Currently, gas chromatography-mass spectrometry (GC-MS) is used in a large number of applications. However, GC requires a certain vapor pressure of the sample, and only 20% of the drug can be satisfactorily separated by gas chromatography without prior chemical treatment, and in many cases the drug under study needs to be properly pretreated and derivatized to make it a sample that is easily vaporized for GC-MS analysis. Meanwhile, the LC-MS online makes up for the defects of the traditional LC detector, has the characteristics of high separation capacity, high sensitivity, wider application range, strong specificity and the like, and is increasingly paid more attention by people. It is estimated that about 80% of the known compounds are all organic compounds with strong hydrophilicity and low volatility, heat labile compounds and biological macromolecules, which are widely existed in the most extensive and potential fields of current application and development, including biological, medical, chemical and environmental aspects, and they need to be separated by LC. Therefore, the combination of LC and MS can solve the problem which cannot be solved by GC-MS.
The liquid chromatogram-mass spectrum combined instrument is a combined instrument of liquid chromatogram and mass spectrum. The method combines the separation capability of a liquid chromatograph for effectively separating heat instability and high-boiling point compounds with the strong component identification capability of a mass spectrometer. Is an effective means for separating and analyzing complex organic mixtures. The key to the on-line process is the development of a suitable interface that must be stripped of solvent before the sample components enter the ion source, and currently, a tracked heated conveyor belt is used in most cases.
Liquid chromatography Mass Spectrometer (liquid chromatography Mass Spectrometer), LC-MS for short, is a high-end instrument in the organic matter analysis market. Liquid Chromatography (LC) can effectively separate organic components in a sample to be tested of organic matters, and Mass Spectrometry (MS) can analyze the separated organic matters one by one to obtain information of molecular weight, structure (in some cases) and concentration (quantitative analysis) of the organic matters. The powerful electrospray ionization technology creates the characteristics of very simple LC-MS mass spectrogram and simple post data processing. LC-MS is an indispensable analytical tool for departments of organic matter analysis laboratories, drug and food inspection rooms, production process control, quality inspection and the like.
The liquid chromatogram-mass spectrum combination instrument is characterized in that: the liquid chromatography is connected with the mass spectrum, so that additional analysis capability can be added, and trace compounds in complex sample matrixes such as cell and tissue lysate, blood, plasma, urine, oral fluid and the like can be accurately identified and quantified. High performance liquid chromatography mass spectrometry systems (abciex eksingent LC/MS and LC/MS) offer some unique advantages, including: (1) minimal sample preparation required for rapid analysis and circulation; (2) high sensitivity in combination with the ability to analyze multiple compounds, even across compound classes; (3) high accuracy, high resolution identification and quantification of target analytes.
Although many analytical chemistry fields have been solved by liquid chromatography-mass spectrometry, the sample for LC-MS analysis is usually required to be an aqueous solution or a methanol solution, and particularly the mobile phase of the liquid chromatography should contain no or as little non-volatile salts as possible.
Unfortunately, as a mobile phase of liquid chromatography such as high performance liquid chromatography, some non-volatile buffer salts are generally required to be added thereto to obtain excellent chromatographic separation effect, but these chromatographic elution fractions containing the non-volatile buffer salts are difficult to remove or separate from the target substances in the chromatographic elution fractions when used in the next mass spectrometric measurement. For example, when measuring an impurity, i.e., a related substance, in aceglutamide contained in the second part, page 6 of the 2010 edition of the chinese pharmacopoeia, separation and analysis are performed by a liquid chromatography using a mobile phase containing potassium dihydrogen phosphate, in which case, if the attribution or chemical structure of an impurity peak is to be determined, it is advantageous to introduce the impurity peak chromatographic fraction into a mass spectrometer for detection, but potassium dihydrogen phosphate contained in the impurity peak chromatographic fraction is not volatile and is not favorable for mass spectrometry. If the peak chromatographic fraction of the impurity is concentrated by the conventional method (usually by nitrogen blowing), although the target substance, i.e., the impurity, is concentrated, the phosphate in the peak chromatographic fraction is also proportionally concentrated, and may cause a change in pH, which affects the stability of the components to be measured, so that the concentration method is not a suitable method for reducing the concentration of the nonvolatile buffer salt in the MS sample. Therefore, the concentration method cannot achieve the purpose of reducing the concentration of the nonvolatile buffer salt in the MS analyte.
Therefore, those skilled in the art of chemical analysis are eagerly expecting a method capable of effectively reducing the content of nonvolatile buffer salt in the test substance of liquid chromatography-mass spectrometry and even removing the nonvolatile buffer salt.
Disclosure of Invention
The application aims to provide a method capable of effectively reducing the content of nonvolatile buffer salt in a test substance of liquid chromatography-mass spectrometry and even removing the nonvolatile buffer salt. It has been surprisingly found that the level of non-volatile buffer salts in a liquid chromatography eluate fraction can be significantly reduced after treatment of the eluate fraction according to the process of the present application. The present application has been completed based on this finding.
To this end, the first method of the present invention provides a method for reducing the content of non-volatile buffer salts in the elution fraction of a liquid chromatography, which method comprises the steps of:
(1) preparing a detection object into a solution, injecting the solution into a liquid chromatograph for elution, and collecting a target object elution fraction to be used for mass spectrum detection from a liquid chromatogram eluent;
(2) thoroughly mixing the elution fraction with an auxiliary (optionally with additional addition of an organic solvent (e.g. methanol and/or acetonitrile));
(3) separating the suspension from the clear liquid by centrifuging or filtering the obtained mixed solution, and separating the clear liquid; and, optionally
(4) Concentrating the obtained clear liquid to obtain the final product.
The method according to any one of the embodiments of the first aspect of the present invention, wherein the liquid chromatography is reverse phase high performance liquid chromatography.
The method according to any one of the embodiments of the first aspect of the present invention, wherein the mobile phase used for the liquid chromatography elution is a mixture of an aqueous phase and an organic phase. In one embodiment, the organic phase is such as, but not limited to: methanol, acetonitrile, ethanol, tetrahydrofuran, isopropanol, and combinations thereof; in particular methanol and acetonitrile. In one embodiment, the aqueous phase comprises a non-volatile buffer salt.
The process according to the present invention according to any one of the embodiments of the first aspect of the present invention, wherein the volume ratio of the aqueous phase and the organic phase may be 5: 95-95: 5 in the above range.
The method according to any embodiment of the first aspect of the present invention, wherein said non-volatile buffer salts are for example but not limited to one or more of phosphates, citrates, acetates, perchlorates.
The method according to any one of the embodiments of the first aspect of the present invention, wherein the base of the non-volatile buffer salt is a base selected from alkali metal or alkaline earth metal ions or is an ammonium group. In one embodiment, the alkali radical is selected from: sodium ion, potassium ion, magnesium ion, calcium ion, preferably the alkali radical is sodium ion, potassium ion, or ammonium ion.
A method according to any embodiment of the first aspect of the invention, wherein said non-volatile buffer salts include, but are not limited to: sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, sodium citrate, potassium citrate, disodium citrate, dipotassium citrate, trisodium citrate, tripotassium citrate, sodium acetate, potassium acetate, ammonium acetate, sodium perchlorate, potassium perchlorate, and combinations thereof.
The method according to any one of the embodiments of the first aspect of the present invention, wherein the target is a major component or a non-major component (e.g. an impurity) in the assay. In one embodiment, the principal or non-principal component is a known substance or an unknown substance. In the field of chemical analysis, after a known substance or an unknown substance is separated in liquid chromatography to obtain a target substance chromatographic peak or an elution fraction, the target substance enters a mass spectrometer for detection, and the chemical structure of the target substance can be determined according to a mass spectrum detection result, so that the known substance can be accurately confirmed, or the chemical structure/composition of the unknown substance can be analyzed.
The method according to any one of the embodiments of the first aspect of the present invention, wherein the solvent used to formulate the test substance into a solution is a mobile phase or a solvent selected from the group consisting of: water, methanol, acetonitrile, ethanol, and combinations thereof.
The process according to any one of the embodiments of the first aspect of the invention, wherein the auxiliary agent is selected from: molecular sieves, silica gels, and combinations thereof.
The process according to any one of the embodiments of the first aspect of the invention, wherein the molecular sieve is sized to haveOf apertures, e.g. havingOf apertures, e.g. havingOf apertures, e.g. havingOrThe pore diameter of (a).
The process according to any one of the embodiments of the first aspect of the present invention, wherein the silica gel is selected from the group consisting of: fine silica gel, coarse silica gel, blue silica gel, allochroic silica gel, cobaltless allochroic silica gel, and combinations thereof.
The process according to any one of the embodiments of the first aspect of the present invention, wherein the elution fraction and the auxiliary agent, when mixed, are present in a weight ratio of 1: 0.01-100, for example, in a weight ratio of 1: 0.05-10, for example, in a weight ratio of 1: 0.1-5, for example, in a weight ratio of 1: 0.5-2. The weight ratio of the two is easily adjusted, for example, the mixing ratio of the elution fraction and the auxiliary is appropriately adjusted according to the degree of reduction of the nonvolatile buffer salt.
The process according to any embodiment of the first aspect of the present invention, wherein the volume ratio of the aqueous phase and the organic phase may be 5: 95-95: 5 in the above range. It has been found that the reduction of the nonvolatile buffer salts used therein can be achieved as long as the detection object and the target object are normally dissolved in this mobile phase. Furthermore, since the method of the first aspect of the invention is particularly useful for LC-MS assays for a certain detection and/or target, the method of the first aspect of the invention is a few simple sample processing steps added in the middle of such an LC-MS method. Thus, it is well understood by those skilled in the art that the LC-MS method suitable for the analysis of analytes and/or targets is well adapted to accept the addition of several process steps of the present invention. That is, it is easy to conceive that the method of the present invention is suitable for any LC-MS method satisfying the measurement conditions of the present invention, and for example, the method of the present invention is suitable for these LC-MSs as long as the mobile phase and buffer salt used in LC of these LC-MSs satisfy the specified conditions. Thus, the detection and target substances in the methods of the invention, and the LC and MS methods used therefor, are not necessarily specifically limited, although the methods of the invention are hereinafter validated as specific drug assays.
According to the method of any one of the embodiments of the first aspect of the present invention, the residual rate (%) of the nonvolatile buffer salt in the clear solution obtained in the step (3) is 50% or less, for example, 5 to 50%, for example, 5 to 40%, for example, 5 to 35%, for example, 5 to 30%, for example, 5 to 25% with respect to the nonvolatile buffer salt in the elution fraction of the target product obtained in the step (1).
According to the method of any one of the embodiments of the first aspect of the present invention, the residual ratio (%) of the target substance in the clear solution obtained in the step (3) with respect to the target substance in the target substance-eluted fraction obtained in the step (1) is 70% or more, for example, 70 to 100%, for example, 70 to 95%, for example, 70 to 90%, for example, 75 to 90%.
The process according to any one of the embodiments of the first aspect of the present invention, wherein the volume of the organic solvent in the step (2) is 0 to 100 times, for example 0 to 50 times, for example 0 to 25 times, for example 0 to 10 times, for example 0 to 5 times the volume of the elution fraction.
Since the auxiliary agents used in the present invention are mostly particles or granules, the separation of suspended matter and clear solution by centrifugation or filtration can easily result in a clear solution suitable for subsequent measurement, which is commonly referred to as clear solution. Thus, for example, for filtration, a common microfiltration membrane can perform these functions, e.g., a 0.45um or 0.22um pore size microfiltration membrane can be used.
Since the target substance fraction obtained by the method of the present invention may be a major component or a trace amount of an impurity component in the analyte, it is necessary to concentrate the supernatant obtained by the method of the present invention, if necessary, particularly if the amount of the impurity is trace, in order to obtain a sufficient concentration of the target substance for the subsequent mass spectrometric measurement. In the case where the target is a main component, the amount of the target contained in the clear solution is large enough for the subsequent mass spectrometric measurement, and therefore the clear solution may not be concentrated.
In general, concentration can be achieved by a method of removing the solvent by evaporation (e.g., nitrogen blow drying). In this concentration case, the elution fraction originating from the mobile phase is not effectively freed from the non-volatile buffer salts which exert a great stress on the subsequent mass spectrometric detection and may cause a change in pH, which affects the stability of the components to be measured. It has been surprisingly found that by the process of the present invention, the level (or concentration) of non-volatile buffer salts in the elution fractions of a liquid chromatography can be effectively reduced.
Further, the second aspect of the present invention provides a method for performing liquid chromatography-mass spectrometry on a target in a test substance, comprising the steps of:
(1) preparing a detection object into a solution, injecting the solution into a liquid chromatograph for elution, and collecting a target object elution fraction to be used for mass spectrum detection from a liquid chromatogram eluent;
(2) thoroughly mixing the elution fraction with an auxiliary (optionally with additional addition of an organic solvent (e.g. methanol and/or acetonitrile));
(3) separating the suspension from the clear liquid by centrifuging or filtering the obtained mixed solution, and separating the clear liquid;
(4) taking the clear liquid obtained in the last step for mass spectrometry directly, or concentrating the clear liquid and then using the concentrated clear liquid for mass spectrometry.
The method according to any one of the embodiments of the second aspect of the present invention, wherein the liquid chromatography is reverse phase high performance liquid chromatography.
The method according to any one of the embodiments of the second aspect of the present invention, wherein the mobile phase used for the liquid chromatography elution is a mixture of an aqueous phase and an organic phase. In one embodiment, the organic phase is such as, but not limited to: methanol, acetonitrile, ethanol, tetrahydrofuran, isopropanol, and combinations thereof; in particular methanol and acetonitrile. In one embodiment, the aqueous phase comprises a non-volatile buffer salt.
The process according to the present invention according to any one of the embodiments of the second aspect of the present invention, wherein the volume ratio of the aqueous phase and the organic phase may be 5: 95-95: 5 in the above range.
The method according to any embodiment of the second aspect of the present invention, wherein said non-volatile buffer salts are for example but not limited to one or more of phosphates, citrates, acetates, perchlorates.
The method according to any one of the embodiments of the second aspect of the present invention, wherein the base of the non-volatile buffer salt is a base selected from alkali metal or alkaline earth metal ions or is an ammonium group. In one embodiment, the alkali radical is selected from: sodium ion, potassium ion, magnesium ion, calcium ion, preferably the alkali radical is sodium ion, potassium ion, or ammonium ion.
A method according to any embodiment of the second aspect of the present invention, wherein said non-volatile buffer salts include, but are not limited to: sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, sodium citrate, potassium citrate, disodium citrate, dipotassium citrate, trisodium citrate, tripotassium citrate, sodium acetate, potassium acetate, ammonium acetate, sodium perchlorate, potassium perchlorate, and combinations thereof.
The method according to any one of the embodiments of the second aspect of the present invention, wherein the target is a major component or a non-major component (e.g. an impurity) in the assay. In one embodiment, the principal or non-principal component is a known substance or an unknown substance. In the field of chemical analysis, after a known substance or an unknown substance is separated in liquid chromatography to obtain a target substance chromatographic peak or an elution fraction, the target substance enters a mass spectrometer for detection, and the chemical structure of the target substance can be determined according to a mass spectrum detection result, so that the known substance can be accurately confirmed, or the chemical structure/composition of the unknown substance can be analyzed.
The method according to any one of the embodiments of the second aspect of the present invention, wherein the solvent used to formulate the test substance into a solution is a mobile phase or a solvent selected from the group consisting of: water, methanol, acetonitrile, ethanol, and combinations thereof.
The process according to any embodiment of the second aspect of the present invention, wherein the auxiliary agent is selected from: molecular sieves, silica gels, and combinations thereof.
The process according to any embodiment of the second aspect of the present invention, wherein the molecular sieve is sized to haveOf apertures, e.g. havingOf apertures, e.g. havingOf apertures, e.g. havingOrThe pore diameter of (a).
A process according to any one of the embodiments of the second aspect of the present invention, wherein the silica gel is selected from: fine silica gel, coarse silica gel, blue silica gel, allochroic silica gel, cobaltless allochroic silica gel, and combinations thereof.
The process according to any one of the embodiments of the second aspect of the present invention, wherein the elution fraction and the auxiliary agent, when mixed, are present in a weight ratio of 1: 0.01-100, for example, in a weight ratio of 1: 0.05-10, for example, in a weight ratio of 1: 0.1-5, for example, in a weight ratio of 1: 0.5-2. The weight ratio of the two is easily adjusted, for example, the mixing ratio of the elution fraction and the auxiliary is appropriately adjusted according to the degree of reduction of the nonvolatile buffer salt.
The process according to any embodiment of the second aspect of the present invention, wherein the volume ratio of the aqueous phase and the organic phase may be 5: 95-95: 5 in the above range.
According to the method of any one of the second aspect of the present invention, the residual (%) of the nonvolatile buffer salt in the clear solution obtained in the step (3) is 50% or less, for example, 5 to 50%, for example, 5 to 40%, for example, 5 to 35%, for example, 5 to 30%, for example, 5 to 25% with respect to the nonvolatile buffer salt in the elution fraction of the target product obtained in the step (1).
According to the method of any one of the embodiments of the second aspect of the present invention, the residual ratio (%) of the target substance in the clear solution obtained in the step (3) with respect to the target substance in the target substance elution fraction obtained in the step (1) is 70% or more, for example, 70 to 100%, for example, 70 to 95%, for example, 70 to 90%, for example, 75 to 90%.
The process according to any one of the embodiments of the first aspect of the present invention, wherein the volume of the organic solvent in the step (2) is 0 to 100 times, for example 0 to 50 times, for example 0 to 25 times, for example 0 to 10 times, for example 0 to 5 times the volume of the elution fraction.
Any embodiment of any aspect of the present application may be combined with other embodiments, as long as they do not contradict. Furthermore, in any embodiment of any aspect of the present application, any feature may be applicable to that feature in other embodiments, as long as they do not contradict.
The present application is further described below.
All documents cited in this application are incorporated herein by reference in their entirety and to the extent that the meaning of such documents is inconsistent with this application, the express disclosure of this application controls. Further, the various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art, and even so, it is intended that the present application be more fully described and interpreted herein, to the extent that such terms and phrases are not inconsistent with this known meaning and from the context in which such terms and phrases are expressed.
Illustratively, in carrying out the present invention, the concentration of the nonvolatile buffer salt in the elution fraction of the target obtained in step (1) of the present invention (which may be referred to as C1 in the present invention and is more convenient in terms of molar concentration, particularly in the case of using a plurality of buffer salts in combination, for example, when using both of monomethyl phosphate and disodium hydrogen phosphate, the total molar concentration of phosphate ions may be determined), followed by the concentration of the nonvolatile buffer salt in the supernatant obtained in step (3) of the present invention (which may be referred to as C2 in the present invention and is the same as C1 in terms of concentration), and then the residual ratio (%) of the buffer salt after the treatment using the method of the present invention may be calculated as follows:
buffer salt residual rate (%) - (C2 ÷ C1) × 100%
It has been found that the residual content of the buffer salt can be reduced to less than 50%, for example to 5 to 40%, for example to 5 to 35%, for example to 5 to 30%, for example to 5 to 25% by the process of the invention. It can be seen that the bulk removal of buffer salts is easily achieved by the simple treatment method of the present invention.
The concentration of non-volatile buffer salts in the target elution fraction and the supernatant can be determined using a number of prior art methods. For example, acetate, phosphate and citrate can be typically determined by reference to the method of Xunming (capillary zone electrophoresis of acetate and phosphate in amino acid injection, J. China pharmaceutical industry, 2015,46(1): 59). As another example, perchlorate is typically measured by reference to the Stahli method (ion chromatography-mass spectrometry, analytical laboratories, 2007,26(4):34), for the determination of trace amounts of perchlorate in drinking water and in environmental water samples. Since the invention uses the relative changes of C1 and C2 to characterize the technical effect of the invention, as long as there is relative comparability between C1 and C2, there is no need for particularly high requirements on the accuracy of the determination of these specific buffer salts. Other assays reported in the literature are also fully applicable.
Illustratively, in carrying out the present invention, the concentration of the target substance in the target substance elution fraction obtained in step (1) of the present invention (which may be referred to as D1 in the present invention, which may be conveniently measured in molar concentration) may be measured, followed by the concentration of the target substance in the supernatant obtained in step (3) of the present invention (which may be referred to as D2 in the present invention, which concentration is expressed in the same manner as D1), and then the remaining rate (%) of the buffer salt after the treatment using the method of the present invention may be calculated as follows:
target residue (%) - (D2 ÷ D1) × 100%
It has been found that the residual ratio (%) of the target product can be 70% or more, for example, 70 to 100%, for example, 70 to 95%, for example, 70 to 90%, for example, 75 to 90% by the method of the present invention.
The method of determining the concentration of the target is readily accomplished based on the assay conditions for the particular product, as will be readily understood in the specific examples below.
The invention adopts a simple processing method, can realize the great reduction of the concentration of the non-volatile buffer salt in the LC elution fraction of the LC-MS method, and can not lose the target substance.
Detailed Description
The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible. The following examples further illustrate the invention without limiting it. Any equivalent changes in form only, but not in material, made in accordance with the present inventive concept should be considered as within the scope of the present invention.
Example 1: method for removing buffer salt sodium dihydrogen phosphate in mobile phase
The second part 130 page of the 2010 edition of the Chinese pharmacopoeia contains captopril and needs to be checked for impurities, namely captopril disulfide. This example examines the effect of removing the buffer salt, sodium dihydrogen phosphate, from the mobile phase.
And (4) avoiding light. Taking a captopril raw material medicine, precisely weighing, adding a mobile phase for dissolving, and quantitatively diluting to prepare a solution containing about 0.5mg in 1ml as a test solution (fresh preparation for clinical use); taking a captopril disulfide reference substance, precisely weighing, adding a proper amount of methanol for dissolving, and quantitatively diluting by using a mobile phase to prepare a solution containing about 5ug of captopril disulfide per 1ml as a reference substance solution; taking the captopril and captopril disulfide reference substance, adding a proper amount of methanol for dissolving, and diluting by using a mobile phase to prepare mixed solution containing 0.1mg and 15ug of each in 1ml as system applicability test solution. Performing high performance liquid chromatography (appendix VD of second part of 2010 edition in Chinese pharmacopoeia) test by using octadecylsilane chemically bonded silica as filler; 0.01mol/L sodium dihydrogen phosphate solution-methanol-acetonitrile (70: 25: 5) (pH adjusted to 3.0 with phosphoric acid) as mobile phase; the detection wavelength is 215 nm; the column temperature was 40 ℃. Taking 50ul of the system applicability test solution, injecting into a liquid chromatograph, wherein the separation degree between the captopril peak and the captopril disulfide peak is more than 4.0. Taking 50ul of the reference solution, injecting into a liquid chromatograph, and adjusting the detection sensitivity to make the peak height of the captopril disulfide chromatographic peak about 50% of the full range; precisely measuring the sample solution and the reference solution at 50ul, respectively, injecting into a liquid chromatograph, and recording chromatogram; if a chromatographic peak consistent with the retention time of captopril disulfide exists in a chromatogram of a test solution, the chromatographic peak is calculated by the peak area according to an external standard method, and the chromatographic peak is not more than 1.0%. From this, the content of captopril disulfide in the drug substance can be determined.
Then, the sample solution was measured precisely 50ul according to the above method, and the sample solution was injected into a liquid chromatograph to cut off the elution fraction (referred to as fraction A herein) of the captopril chromatographic peak portion and the elution fraction (referred to as fraction B herein) of the captopril disulfide chromatographic peak portion, respectively. For fraction a, the captopril concentration therein (denoted D1) and the phosphate concentration therein (denoted C1) were determined. The captopril disulfide concentration in the elution fraction of the chromatographic peak portion of captopril disulfide and the phosphate concentration in the elution fraction can also be similarly determined.
Subsequently, fraction A or fraction B, respectively, is treated according to the following process steps of the invention:
(1) preparing a detection object into a solution, injecting the solution into a liquid chromatograph for elution, and collecting a target object elution fraction to be used for mass spectrum detection from a liquid chromatogram eluent (the step is completed, and the obtained fractions are fraction A or fraction B respectively);
(2) the elution fraction is combined with an auxiliary (molecular sieve,elution fraction and weight of auxiliaryAnd (3) a ratio of about 1: 1) fully mixing;
(3) separating the suspension from the clear liquid by centrifuging the obtained mixed solution, and separating the clear liquid; measuring the captopril concentration or captopril disulfide concentration (recorded as D2) in the clear liquid, and the phosphate concentration (recorded as C2) in the clear liquid;
(4) directly using the captopril clear liquid obtained in the last step for mass spectrometry to determine whether the captopril clear liquid is anastomotic with the captopril; the resulting clear captopril disulfide solution was concentrated by nitrogen blow-drying (to 1/10 volumes) and then used for mass spectrometry to determine whether it coincided with the captopril disulfide. (in the case where the sample amount is insufficient at the time of MS measurement, the step (1) may be repeated to obtain a plurality of elution fractions and combine them, the same applies hereinafter).
Example 2: method for removing buffer salt sodium dihydrogen phosphate in mobile phase
Fraction A or fraction B obtained in this way were treated according to example 1 according to the following procedure of the process of the invention:
(1) preparing a detection object into a solution, injecting the solution into a liquid chromatograph for elution, and collecting a target object elution fraction to be used for mass spectrum detection from a liquid chromatogram eluent (the step is completed, and the obtained fractions are fraction A or fraction B respectively);
(2) the elution fraction is combined with an auxiliary (molecular sieve,elution fraction to adjuvant weight ratio about 1: 0.5) fully mixing;
(3) separating the suspension from the clear liquid by filtering (microporous membrane, 0.45um) the obtained mixed solution, and separating the clear liquid; measuring the captopril concentration or captopril disulfide concentration (recorded as D2) in the clear liquid, and the phosphate concentration (recorded as C2) in the clear liquid;
(4) directly using the captopril clear liquid obtained in the last step for mass spectrometry to determine whether the captopril clear liquid is anastomotic with the captopril; the resulting clear captopril disulfide solution was concentrated by nitrogen blow-drying (to 1/5 volumes) and then used for mass spectrometry to determine whether it coincided with the captopril disulfide.
Example 3: method for removing buffer salt sodium dihydrogen phosphate in mobile phase
Fraction A or fraction B obtained in this way were treated according to example 1 according to the following procedure of the process of the invention:
(1) preparing a detection object into a solution, injecting the solution into a liquid chromatograph for elution, and collecting a target object elution fraction to be used for mass spectrum detection from a liquid chromatogram eluent (the step is completed, and the obtained fractions are fraction A or fraction B respectively);
(2) the elution fraction is combined with an auxiliary (molecular sieve,elution fraction to adjuvant weight ratio about 1: 2) mixing thoroughly, adding acetonitrile (25 times of the volume of the eluted fraction), and mixing thoroughly;
(3) separating the suspension from the clear liquid by filtering (microporous membrane, 0.22um) the obtained mixed solution, and separating the clear liquid; measuring the captopril concentration or captopril disulfide concentration (recorded as D2) in the clear liquid, and the phosphate concentration (recorded as C2) in the clear liquid;
(4) directly using the captopril clear liquid obtained in the last step for mass spectrometry to determine whether the captopril clear liquid is anastomotic with the captopril; the resulting clear captopril disulfide solution was concentrated by nitrogen blow-drying (to 1/5 volumes) and then used for mass spectrometry to determine whether it coincided with the captopril disulfide.
Example 4: removal of buffer salt phosphoric acid in mobile phaseProcess for preparing sodium dihydrogen
Fraction A or fraction B obtained in this way were treated according to example 1 according to the following procedure of the process of the invention:
(1) preparing a detection object into a solution, injecting the solution into a liquid chromatograph for elution, and collecting a target object elution fraction to be used for mass spectrum detection from a liquid chromatogram eluent (the step is completed, and the obtained fractions are fraction A or fraction B respectively);
(2) the elution fraction is combined with an auxiliary (molecular sieve,elution fraction to adjuvant weight ratio about 1: 1) mixing thoroughly, adding acetonitrile (10 times of the volume of the eluted fraction), and mixing thoroughly;
(3) separating the suspension from the clear liquid by filtering (microporous membrane, 0.45um) the obtained mixed solution, and separating the clear liquid; measuring the captopril concentration or captopril disulfide concentration (recorded as D2) in the clear liquid, and the phosphate concentration (recorded as C2) in the clear liquid;
(4) directly using the captopril clear liquid obtained in the last step for mass spectrometry to determine whether the captopril clear liquid is anastomotic with the captopril; the resulting clear captopril disulfide solution was concentrated by nitrogen blow-drying (to 1/5 volumes) and then used for mass spectrometry to determine whether it coincided with the captopril disulfide.
Example 5: method for removing buffer salt sodium dihydrogen phosphate in mobile phase
Fraction A or fraction B obtained in this way were treated according to example 1 according to the following procedure of the process of the invention:
(1) preparing a detection object into a solution, injecting the solution into a liquid chromatograph for elution, and collecting a target object elution fraction to be used for mass spectrum detection from a liquid chromatogram eluent (the step is completed, and the obtained fractions are fraction A or fraction B respectively);
(2) thoroughly mixing the elution fraction with an auxiliary (fine-pored silica gel, elution fraction to auxiliary in a weight ratio of about 1: 0.5);
(3) separating the suspension from the clear liquid by filtering (microporous membrane, 0.22um) the obtained mixed solution, and separating the clear liquid; measuring the captopril concentration or captopril disulfide concentration (recorded as D2) in the clear liquid, and the phosphate concentration (recorded as C2) in the clear liquid;
(4) directly using the captopril clear liquid obtained in the last step for mass spectrometry to determine whether the captopril clear liquid is anastomotic with the captopril; the resulting clear captopril disulfide solution was concentrated by nitrogen blow-drying (to 1/10 volumes) and then used for mass spectrometry to determine whether it coincided with the captopril disulfide.
Example 6: method for removing buffer salt sodium dihydrogen phosphate in mobile phase
Fraction A or fraction B obtained in this way were treated according to example 1 according to the following procedure of the process of the invention:
(1) preparing a detection object into a solution, injecting the solution into a liquid chromatograph for elution, and collecting a target object elution fraction to be used for mass spectrum detection from a liquid chromatogram eluent (the step is completed, and the obtained fractions are fraction A or fraction B respectively);
(2) mixing the elution fraction with auxiliary agent (allochroic silicagel, the weight ratio of elution fraction to auxiliary agent is about 1: 2), adding methanol (the volume of methanol is 50 times of that of elution fraction), and mixing;
(3) separating the suspension from the clear liquid by filtering (microporous membrane, 0.22um) the obtained mixed solution, and separating the clear liquid; measuring the captopril concentration or captopril disulfide concentration (recorded as D2) in the clear liquid, and the phosphate concentration (recorded as C2) in the clear liquid;
(4) directly using the captopril clear liquid obtained in the last step for mass spectrometry to determine whether the captopril clear liquid is anastomotic with the captopril; the resulting clear captopril disulfide solution was concentrated by nitrogen blow-drying (to 1/10 volumes) and then used for mass spectrometry to determine whether it coincided with the captopril disulfide.
Example 7: method for removing buffer salt sodium dihydrogen phosphate in mobile phase
Fraction A or fraction B obtained in this way were treated according to example 1 according to the following procedure of the process of the invention:
(1) preparing a detection object into a solution, injecting the solution into a liquid chromatograph for elution, and collecting a target object elution fraction to be used for mass spectrum detection from a liquid chromatogram eluent (the step is completed, and the obtained fractions are fraction A or fraction B respectively);
(2) thoroughly mixing the elution fraction with an auxiliary agent (cobalt-free allochroic silica gel, the weight ratio of the elution fraction to the auxiliary agent is about 1: 0.5);
(3) separating the suspension from the clear liquid by filtering (microporous membrane, 0.22um) the obtained mixed solution, and separating the clear liquid; measuring the captopril concentration or captopril disulfide concentration (recorded as D2) in the clear liquid, and the phosphate concentration (recorded as C2) in the clear liquid;
(4) directly using the captopril clear liquid obtained in the last step for mass spectrometry to determine whether the captopril clear liquid is anastomotic with the captopril; the resulting clear captopril disulfide solution was concentrated by nitrogen blow-drying (to 1/10 volumes) and then used for mass spectrometry to determine whether it coincided with the captopril disulfide.
As a result: fractions A and B were determined by mass spectrometry from examples 1-7 above, and the mass spectrometry results in each run were consistent with captopril and captopril disulfide, respectively; in each test, the residual rate of the buffer salt was in the range of 6 to 24%, for example, the residual rate of the buffer salt in example 1 was 14%; the captopril target residue ranges from 71% to 86%, for example 78% for the captopril target of example 2; the captopril disulfide target residue ranges from 73% to 84%, for example 79% for the captopril disulfide target of example 3.
Example 8: method for removing mixed buffer salts of potassium dihydrogen phosphate and disodium hydrogen phosphate in mobile phase
The second part 179 of the chinese pharmacopoeia 2010 edition contains a method for determining the content of a cefodizime sodium raw material drug by using an HPLC method, and the effect of removing mixed buffer salts in a mobile phase is examined in this example.
Octadecylsilane chemically bonded silica is used as a filling agent; taking phosphate buffer solution (taking 0.87g of monopotassium phosphate and 0.22g of anhydrous disodium hydrogen phosphate, adding water to dissolve and dilute the phosphate buffer solution to 1000ml, shaking up) -acetonitrile (920: 80) as a mobile phase; the detection wavelength was 262 nm. Taking 10ml of reference solution, adding 1ml of 0.1mol/L hydrochloric acid solution, standing at room temperature for 24 hours, adding 1ml of 0.1mol/L sodium hydroxide solution, shaking up, taking 20ul, and injecting into a liquid chromatograph, wherein the separation degrees of the craw peak of cefdinir and the adjacent degradation impurity peaks before and after the craw peak of cefdinir are respectively not less than 3.0 and 4.0. The determination method comprises the following steps: taking a proper amount of the product, precisely weighing, adding water to dissolve, quantitatively diluting to prepare a solution containing about 0.1mg of cefodizime in every 1ml, shaking uniformly, precisely weighing 20ul, injecting into a liquid chromatograph, and recording a chromatogram; taking another appropriate amount of cefodizime reference substance, and determining by the same method. The content of the test sample C20H20N6O7S4 is calculated by the external standard method according to the peak area. The method can determine the content of cefodizime C20H20N6O7S4 in the bulk drug test sample.
Then, as described above, 20ul of the sample solution was measured again, and the sample solution was injected into a liquid chromatograph to intercept the elution fraction (referred to as fraction A herein) at the peak of the cefodizime chromatogram. The fraction A was measured for its cefodizime concentration (designated D1) and for its phosphate concentration (designated C1).
Next, the above fraction A was treated in the same manner as in the steps (1) to (4) in examples 1 to 7, respectively.
As a result: mass spectrum results are matched with cefodizime through mass spectrum measurement; the residual rate of the buffer salt is in the range of 6-23%, and the residual rate of the target substance is in the range of 73-90%.
Example 9: method for removing mixed buffer salt acetate and perchlorate in mobile phase
The second part 107 of the 2010 edition of the Chinese pharmacopoeia contains a method for measuring the content of levofloxacin raw material medicines by using an HPLC method, and the effect of removing acetate and perchlorate of mixed buffer salt in a mobile phase is examined in the example.
Octadecylsilane chemically bonded silica is used as a filling agent; dissolving ammonium acetate sodium perchlorate solution (4.0 g ammonium acetate and 7.0g sodium perchlorate in 1300ml water), adjusting pH to 2.2 with phosphoric acid) -acetonitrile (85: 15) as mobile phase; the detection wavelength was 294 nm. Weighing appropriate amount of each of the levofloxacin reference substance, the ciprofloxacin reference substance and the impurity E reference substance, adding 0.1mol/L hydrochloric acid solution to dissolve and dilute the solution to prepare a mixed solution containing 0.12mg of levofloxacin, 6ug of ciprofloxacin and 6ug of impurity E in each 1ml of solution, injecting 10ul of the mixed solution into a liquid chromatograph, recording a chromatogram, wherein the retention time of a levofloxacin peak is about 15 minutes, and the separation degrees of the levofloxacin peak and the impurity E peak and the separation degrees of the levofloxacin peak and the ciprofloxacin peak are respectively more than 2.0 and 2.5. Precisely weighing about 60mg of levofloxacin raw material, placing the levofloxacin raw material into a 50ml measuring flask, adding 0.1mol/L hydrochloric acid solution for dissolving and quantitatively diluting to a scale, shaking up, precisely weighing 5ml, placing the levofloxacin raw material into a 50ml measuring flask, diluting to a scale by using 0.1mol/L hydrochloric acid solution, shaking up, precisely weighing 10ul, injecting into a liquid chromatograph, and recording a chromatogram; and taking an appropriate amount of levofloxacin reference substances, measuring by the same method, and calculating the levofloxacin content by peak area according to an external standard method to obtain the levofloxacin reference substance. The method can determine the content of levofloxacin in the crude drug test sample.
Then, 20ul of the sample solution was measured again precisely in the same manner as described above, and the sample solution was injected into a liquid chromatograph to intercept the elution fraction (herein, referred to as fraction A) of the levofloxacin peak portion. The fraction a was measured for its levofloxacin concentration (designated D1) and for its acetate concentration (designated C1) and perchlorate concentration (designated C2).
Next, the fraction A was treated in accordance with the procedures of steps (1) to (4) in examples 1, 3, 5 and 6, respectively.
As a result: the mass spectrum result is matched with levofloxacin through mass spectrum measurement; the residual rate of acetate is within the range of 8-21%, the residual rate of perchlorate is within the range of 10-23%, and the residual rate of target substances is within the range of 74-89%.
Example 10: method for removing mixed buffer salt acetate and perchlorate in mobile phase
The second part of the Chinese pharmacopoeia, 2010 edition, 221, contains a method for determining the content of a sparfloxacin raw material drug by using an HPLC method, and the effect of removing a buffer salt citrate in a mobile phase is examined in the example.
Octadecylsilane chemically bonded silica is used as a filling agent; adding sodium citrate buffer solution (weighing citric acid 2.104g and sodium citrate 2.941g, adding water to 500ml, adjusting pH to 2.4 with 70% perchloric acid solution) -acetonitrile (70: 30) as mobile phase; the detection wavelength was 298 nm. Taking a proper amount of sparfloxacin reference substance, adding a mobile phase for dissolving and diluting to prepare a solution containing about 0.1mg per 1ml, irradiating for 20 hours under the illumination of 4500lx to serve as a system applicability test solution, measuring 10ul of a human-injected liquid chromatograph, recording a chromatogram, wherein the retention time of a sparfloxacin peak is about 7 minutes, and the separation degree of the sparfloxacin peak and an impurity peak at which the relative retention time is about 0.9 meets the requirement.
Taking about 50mg of sparfloxacin raw material, precisely weighing, placing in a 100ml measuring flask, adding an appropriate amount of methanol, fully shaking to dissolve, diluting to a scale with methanol, shaking up, precisely measuring 2ml, placing in a 25ml measuring flask, diluting to a scale with a mobile phase, shaking up, precisely measuring 20ul of a human-injected liquid chromatograph, and recording a chromatogram; taking another appropriate amount of sparfloxacin reference substance, measuring by the same method, and calculating by peak area according to an external standard method to obtain the final product. The method can determine the content of the sparfloxacin in the bulk drug test sample.
Then, in the same manner as described above, 20ul of the sample solution was measured again, and the sample solution was injected into a liquid chromatograph to cut the elution fraction (referred to as fraction A herein) of the sparfloxacin peak. The concentration of sparfloxacin (marked as D1) and the concentration of citrate therein (marked as C1) in fraction A were measured.
Next, the fraction A was treated in accordance with the procedures of steps (1) to (4) in examples 1, 3, 5 and 6, respectively.
As a result: the mass spectrum result is matched with the sparfloxacin through mass spectrum determination; the residual rate of citrate is within the range of 6-18%, and the residual rate of the target substance is within the range of 73% -88%.
The present invention, through the above exemplary experiments, can achieve a substantial reduction in the concentration of non-volatile buffer salts in the LC elution fraction of the LC-MS process without loss of target material, using a simple processing method. This is extremely beneficial for the latter MS assay.
While the present application has been described in detail with respect to the various aspects thereof, it is not intended that the present application be limited to the specific embodiments described herein, but rather that the spirit and scope of the present application be limited only by the terms of the appended claims.
Claims (4)
1. A method for reducing the content of non-volatile buffer salts in elution fractions of liquid chromatography in reversed-phase high performance liquid chromatography-mass spectrometry analysis tests comprises the following steps:
(1) preparing a detection object into a solution, injecting the solution into a liquid chromatograph for elution, and collecting a target object elution fraction to be used for mass spectrum detection from a liquid chromatogram eluent; the mobile phase used for the liquid chromatography elution is a mixed solution of an aqueous phase and an organic phase, and the aqueous phase contains non-volatile buffer salt;
(2) contacting the eluted fraction withFully mixing an auxiliary agent and an optional organic solvent acetonitrile, wherein the auxiliary agent is selected from the following components: having a pore diameter ofThe elution fraction and the auxiliary agent are mixed according to the weight ratio of 1: mixing at a ratio of 0.1-5;
(3) separating the suspension from the clear liquid by centrifuging or filtering the obtained mixed solution, and separating the clear liquid; and, optionally
(4) Concentrating the obtained clear liquid to obtain the final product,
wherein,
the non-volatile buffer salt is sodium dihydrogen phosphate,
the target is selected from: captopril, captopril disulfide, or a combination thereof,
the organic phase is selected from: methanol, acetonitrile, and combinations thereof, and the volume ratio of the aqueous phase to the organic phase is 5: 95-95: in the range of 5, the content of the compound,
the volume of the organic solvent in the step (2) is 0-10 times of the volume of the elution fraction,
the residual rate of the nonvolatile buffer salt in the clear solution obtained in the step (3) is about 5 to 25% with respect to the nonvolatile buffer salt in the target elution fraction obtained in the step (1),
and (3) the residual rate of the target object in the clear liquid obtained in the step (3) is 75-90% relative to the target object in the target object elution fraction obtained in the step (1).
2. The method according to claim 1, wherein the color-changing silica gel is selected from the group consisting of blue silica gel and cobalt-free color-changing silica gel.
3. The process according to claim 1, the elution fraction and the auxiliary agent, when mixed, being present in a weight ratio of 1: 0.5-2.
4. The process according to claim 1, wherein the volume of the organic solvent in the step (2) is 0 to 5 times the volume of the eluted fraction.
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